Hedgehog (Hh) signaling pathway is a pathway that is ancient and in highly conservative development, and it participates in the embryo formation of most tissues and maintains and repairs mature tissues in the adult body. The members of the hedgehog family in secreted proteins control cell proliferation, differentiation and tissue formation. Hh signal in the adult body is significantly reduced as compared with those in embryo and newborn body. Inappropriate Hh pathway is activated in certain types of cancers. The cancers include, for example, basal cell carcinoma (Ervin H. Epstein (2008) Nat Rev Cancer. 8: 743-54), medulloblastoma (Raffel, C. (1997) Cancer Research. 57: 842-845), small cell lung cancer (Watkins, D. N. et al (2003) Nature 422, 313-317), pancreatic cancer (Thayer, S. P. et al. (2003) Nature 425, 851-856), prostate tumor (Karhadkar, S. S., et al. (2004) Nature 431, 707-712), and chronic lymphocytic leukemia (Hegde, C. V. et al (2008) Mol Cancer Res. 6 (12): 1928-36). Therefore, the inhibition of abnormal Hh signaling pathway is a remarkable target to the design of anticancer drug.
Hedgehog gene was first identified in Drosophila. The mutation of Hh results in abnormal spike covering the back of the larvae, developing to prompt Hedgehog (Nusslein-Volhard, C. et al. 1980, Nature 287, 795-801). In mammalian cells, three Hedgehog genes, Sonic hedgehog (Shh), Indian hedgehog (Ihh) and Desert hedgehog (Dhh) have been identified (Echelard, Y. et al; Cell 1993, 75, 1417-1430). Shh is the most common hedgehog member in mammals, and is also the preferably characterized ligand in hedgehog family. Prior to secretion, Shh undergoes intramolecular cleavage and lipid modification reaction. The lipid-modified peptide is the cause for the activation of all signals. Two transmembrane proteins are involved in signal transduction of Hh pathway: twelve-transmembrane Patched receptor (PTCH), and seven-transmembrane Smoothened protein (Smo), which act as important positive medium for Hh signaling. The three Hh proteins are used as ligands to start Hh signaling. The receptor of Hh pathway is Ptch, twelve-pass transmembrane protein). When lacking Hh ligand, Ptch binds G-protein that is coupled with receptor-like signal transduction smoothing factor (Smo), and blocks its function (Eggenschwiler, J. T. (2007) Annu. Rev. Cell Dev. Biol. 23, 345-373). The binding with Hh ligand reduces the inhibitory effect of Ptch-mediated Smo. Upon binding Hh ligand, the inhibition of Ptch on Smo is reduced. Then, Smo is activated, and starts signaling cascade, the signaling cascade leading to the activation of inscriptional factors Gli1-3 (Alexandre et al (1996) Genes Dev. 10: 2003-13), which regulates Hh target genes including cyclin D, cyclin E (Duman-Scheel, M. et al (2002) Nature 417, 299-304), c-myc (Ingham P W et al. Genes Dev 2001; 15(23): 3059-87) and Bcl2 (Regl, G. et al. (2004) Cancer Res. 64, 7724-7731). The pathway is an important regulator of cell cycle and differentiation during development.
In the past decade, it is increasingly evident that the abnormal expression of Hh pathway members will lead to the formation and maintenance of cancer. It has been reported that a number of genes (for example, Shh, PTCH1, Smo and Gli) promote the development of different cancers. PTCH1 is the first Hh network gene associated with cancer. The patient with genetic negative mutant PTCH (which may cause constitutive activation of Hh signaling) has a high incidence of basal cell carcinoma and medulloblastoma (Johnson, R. L. et al (1996) Science 272, 1668-1671, Epstein, E. H. (2008) Nat. Rev. Cancer 8, 743-754). The haploinsufficiency of the two genes PTCH and SUFU is associated with rhabdomyosarcoma especially fetal rhabdomyoma and embryonic rhabdomyosarcoma (Tostar, U. et al. (2006), J. Pathol. 208, 17-25). The mutation of Smo or Ptch1 in cerebellar neurons, which leads to constitutive activation of Smo, usually may cause medulloblastoma (Vorechovsky, I. et al. (1997) Oncogene 15, 361-366). It has been found that, compared to that in healthy cells, the transcriptional level of Smo, Gli1 and Ptch1 in oncogenic multiple myeloma cells upregulates significantly. Because Hh pathway is generally involved in cancer, the method of blocking this pathway is highly interesting. It has been shown that the inhibition of the Hedgehog pathway activity with small molecules will cause cell death in many different types of cancers with uncontrolled hedgehog pathway activation.
Among hedgehog pathway inhibitors, cyclopamine (a plant veratrine) is quite interesting. Cyclopamine directly binds Smo, and acts as an antagonist of the Hh pathway. Animal trials verified that cyclopamine exhibited anti-tumor activity in a number of models of small cell lung cancer, medulloblastoma, prostate cancer and gastrointestinal cancer. Despite of its attractive pharmacological properties, the use of cyclopamine for systemic treatment may be subjected to the following restrictions: 1) teratogenic, including: loss of midline facial features, uniocular, lack of anatomical features from forebrain; 2) poorly water-soluble (5 μg/mL); and 3) chemically instable at low pH (Chen. J. K., al. (2002), Proc. Natl. Acad. Sci. U.S.A, 99, 14071-14076).